Bioinformatics analysis of miRNA

What is already known on this topic? MicroRNAs are oligonucleotide sequences that exert a specific biological function. What does this study add? New Evidence about insights into miRNA expression patterns. Give an important highlight of relation to be considered a biological marker for T1D. And can be used, for certain studied miRNA molecules, as highly predictive for disease-associated glycemic derangements depending on the reliability of the used methodology.

Polymorphisms of microRNA (miRNA), as a novel mechanism, are closely associated with disease states by interfering with miRNA function. Direct correlations have been identified between single-nucleotide polymorphisms (SNPs) in miRNA, but the effect on type 2 diabetes mellitus (T2DM) onset among Chinese population remains unclear. Therefore, the aim of this study was to identify correlations between common SNPs in miR-27a, miR-146a, and miR-124a with T2DM among a Chinese population, as well as to explore diabetic pathological mechanisms and the impact of environmental factors. SNPscan technology was used to genotype 995 patients newly diagnosed with T2DM and 967 controls. Logistic regression analysis was performed to compare mutation frequencies between cases and controls. We found no significant correlations between all genotypes of these miRNAs and T2DM in our research. However, stratification analysis identified a lower risk of T2DM associated with the rs531564GC genotype among yo...

The conserved TRIM-NHL protein, NHL-2, plays a key role in small RNA pathways in Caenorhabditis elegans. NHL-2 has been shown to interact with U-rich RNA through its NHL domain, but the importance to its biological function is unknown. We defined the crystal structure of the NHL domain to 1.4 Å resolution and identified residues that affect affinity for U-rich RNA. Functional analysis of an NHL-2 RNA-binding loss-of-function mutant demonstrated defects in the heterochronic pathway, suggesting that RNA binding is essential for its role in this miRNA pathway. Processing bodies were enlarged in the NHL-2 RNA-binding mutant, suggesting a defect in mRNA decay. We also identified the eIF4E binding protein IFET-1 as a strong synthetic interactor with NHL-2 and the DEAD box RNA helicase CGH-1 (DDX6), linking NHL-2 function to translation repression. We demonstrated that in the absence of NHL-2, there was an enrichment of miRNA transcripts associated with the miRNA pathway Argonaute proteins ALG-2 and ALG-2. We demonstrate that NHL-2 RNA-binding activity is essential for let-7 family miRNA-mediated translational repression. We conclude that the NHL-2, CGH-1, and IFET-1 regulatory axes work with the core miRISC components to form an effector complex that is required for some, but not all, miRNAs.

In the present study, we used high-throughput sequencing, miRNA-seq, to discover and explore the expression profiles of known and novel miRNAs in TLR ligand-stimulated vis-àvis non-stimulated (i.e. Control) peripheral blood mononuclear cells (PBMCs) isolated from blood of healthy Murrah buffaloes. Six small RNA (sRNA) libraries were multiplexed in Ion Torrent PI chip and sequenced on Ion Proton System. The reads obtained were aligned to the Bos taurus genome (UMD3.1 assembly), which is phylogenetically closest species to buffalo (Bubalus bubalis). A total of 160 bovine miRNAs were biocomputationally identified in buffalo PBMCs and 130 putatively novel miRNAs (not enlisted in the bovine mirBase) were identified. All of these 290 miRNAs identified across the six treatment and control samples represent the repertoire of novel miRNAs for the buffalo species. The expression profiles of these miRNAs across the samples have been represented by sample dendrogram and heatmap plots. The uniquely expressed miRNAs in each treatment and control groups were identified. A few miRNAs were expressed at very high levels while the majority of them were moderately expressed. The miRNAs bta-miR-103 and -191 were found to be highly abundant and expressed in all the samples. Other abundantly expressed miRNAs include bta-miR-19b, -29b, -15a, -19a, -30d, -30b-5p and members of let family (let 7a-5p, let 7g & let 7f) in in Poly I:C stimulated PBMCs. Only one novel miRNA (bta-miR-11039) out of 130 identified putatively novel miR-NAs, was expressed in all the six samples and differentially expressed (>2-fold) miRNAs were identified. Six of the differentially expressed miRNAs across the groups (bta-miR-421, bta-let-7i, bta-miR-138, bta-miR-21-5p, bta-miR-222 and bta-miR-27b) were subsequently confirmed by TaqMan quantitative reverse transcription polymerase chain reaction (qRT-PCR). Furthermore, the target genes of differentially expressed miRNAs were enriched for the roles in innate immunity and TLR signaling pathways. This maiden study on profiling and cataloguing of bubaline miRNAs expressed in TLR-ligand stimulated PBMCs will

According to findings, long non-coding RNAs (lncRNAs) have an important function in the onset and growth of various cancers, including rectal cancer (RC). RC offers unique issues in terms of diagnosis, treatment, and results, needing a full understanding of the cellular mechanisms that cause it to develop. This thorough study digs into the various functions that lncRNAs perform in RC, giving views into their multiple roles as well as possible therapeutic consequences. The function of lncRNAs in RC cell proliferation, apoptosis, migratory and infiltrating capacities, epithelial-mesenchymal shift, and therapy tolerance are discussed. Various lncRNA regulatory roles are investigated in depth, yielding information on their effect on essential cell functions such as angiogenesis, death, immunity, and growth. Systemic lncRNAs are currently acknowledged as potential indications for the initial stages of identification of cancer, with the ability to diagnose as well as forecast. Besides adding to their diagnostic utility, lncRNAs offer therapeutic opportunities as actors, contributing to the expanding landscape of cancer research. Moreover, the investigation looks into the assessment and predictive utility of lncRNAs as RC markers. The article also offers insight into lncRNAs as chemoresistance and drug resistance facilitators in the setting of RC.

The discovery of microRNAs (miRNAs) has introduced a new paradigm into gene regulatory systems. Large numbers of miRNAs have been identified in a wide range of species, and most of them are known to downregulate translation of messenger RNAs (mRNAs) via imperfect binding of the miRNA to a specific site or sites in the 3 0 untranslated region (UTR) of the mRNA. Identification of genes targeted by miRNAs is widely believed to be an important step toward understanding the role of miRNAs in gene regulatory networks. As part of the effort to understand interactions between miRNAs and their targets, computational algorithms have been developed based on observed rules for features such as the degree of hybridization between the two RNA molecules. These in silico approaches provide important tools for miRNA target detection, and together with experimental validation, help to reveal regulated targets of miRNAs. Here, we summarize the knowledge that has been accumulated about the principles of target recognition by miRNAs and the currently available computational methodologies for prediction of miRNA target genes.

Background

miRNAs are endogenous, non-coding and evolutionarily conserved RNA molecules. They have been found to be involved in the progression and proliferation of various cancers due to their contribution in post-transcriptional regulation. Stomach Adenocarcinoma (STAD) and Liver Hepatocellular Carcinoma (LIHC) are the two most common cancers of the upper intestinal tract. Our study aimed to evaluate the circulating miRNAs from both STAD and LIHC samples and to identify commonly dysregulated miRNAs as biomarkers to detect both cancers at the same time.

Methods and materials

Common differentially expressed miRNAs (DEMs) from GEO and Bioexpress datasets were considered for initial processing in the analysis. Pathway analysis of the selected miRNAs through DIANA-miRpath tool, followed by survival analysis based on prognostic values through OncoLnc server led to the final biomarker candidates for diagnosis and prognosis of STAD and LIHC. An elaborate miRNA-gene-cancer network was set up for a specialized understanding of the selected DEMs corresponding to the specifically unique target genes and the cancer types. The gene ontology analysis was performed using BINGO to determine functional connotations of the differentially expressed genes (DEGs).

Results

After a thorough analysis, we found that the 4 miRNAs: miR-183-5p, miR-203-3p, miR-126-3p and miR-25-3p could be potential prognostic biomarkers against both STAD and LIHC. The differentially expressed genes (DEGs) for these miRNAs were inferred through GEPIA and miRwalk v2.0. The miRNA-gene-cancer network revealed that the commonly deregulated miRNAs could influence the same genes and pathways altered by multiple cancers at the same time-in our case, STAD and LIHC. To support our claim, we showed the gene ontology analysis by BINGO, attesting the functional assignment of the DEGs behind metastasis and development of both the cancers.

Conclusion

Our study evaluated a particularly effective avenue of identifying novel miRNA for both early diagnostic and prognostic purposes against more than one cancer.

ABSTRACTASNA1 plays an essential role in cisplatin chemotherapy response, type 2 diabetes, and heart disease. It is also an important biomarker in the treatment response of many diseases. Biochemically, ASNA1 has two mutually exclusive redox modulated roles: a tail-anchored protein (TAP) targeting function in the reduced state and a holdase/chaperone function in the oxidized state. Assigning biochemical roles of ASNA-1 to biomedical functions is crucial for successful therapy development. Our previous work showed the relevance of the C. elegans ASNA-1 homolog in modeling cisplatin response and insulin secretion. Here we analyzed two-point mutants in highly conserved residues in C. elegans ASNA-1 and identified their importance in separating cisplatin response from its roles in insulin secretion. Further, using targeted depletion we showed asna-1 tissue requirements for C. elegans growth and development. We concluded that, targeting single residues in ASNA-1 affecting Switch I/II dom...

Background/Aim: Several links between DNA replication, pluripotency and development have been recently identified. The involvement of miRNA in the regulation of cell cycle events and pluripotency factors has also gained attention. Materials and Methods: In the present study, we used the g:Profiler platform to analyze transcription factor binding sites, miRNA networks and protein-protein interactions to identify novel links among the aforementioned processes. Results and Conclusion: A complex circuitry between retinoic acid signaling, SWI/SNF components, pluripotency factors including Oct4, Sox2 and Nanog and cell cycle regulators was identified. It is suggested that the DNA replication inhibitor geminin plays a central role in this circuitry.

This study aimed to optimise techniques for whole transcriptome and small RNA analyses on clinical tissue samples from patients with cardiovascular disease. Clinical samples often represent a particular challenge to extracting RNA of sufficient quality for robust RNA sequencing analysis, and due to availability, it is rarely possible to optimise techniques on the samples themselves. Therefore, we have used equivalent samples from pigs undergoing cardiopulmonary bypass surgery to test different protocols for optimal RNA extraction, and then validated the protocols in human samples. Here we present an assessment of the quality and quantity of RNA obtained using a variety of commercially-available RNA extraction kits on both left ventricular biopsies and blood plasma. RNA extraction from these samples presents different difficulties; left ventricular biopsies are small and fibrous, while blood plasma has a low RNA content. We have validated our optimised extraction techniques on human clinical samples collected as part of the ARCADIA (Association of non-coding RNAs with Coronary Artery Disease and type 2 Diabetes) cohort study, resulting in successful whole transcriptome and small RNA sequencing of human left ventricular tissue.

Astrocytes actively regulate numerous cell types both within and outside of the central nervous system in health and disease. Indeed, astrocyte morphology, gene expression and function, alongside the content of astrocyte-derived extracellular vesicles (ADEVs), is significantly altered by ageing, inflammatory processes and in neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease and amyotrophic lateral sclerosis. Here, we review the relevant emerging literature focussed on perturbation in expression of microRNA (miRNA), small non-coding RNAs that potently regulate gene expression. Synthesis of this literature shows that ageing-related processes, neurodegenerative disease-associated mutations or peptides and cytokines induce dysregulated expression of miRNA in astrocytes and in some cases can lead to selective incorporation of miRNA into ADEVs. Analysis of the miRNA targets shows that the resulting downstream consequences of alterations to levels of miRNA includ...

MicroRNAs (miRNAs) are regulatory small non-coding RNAs that function as translational repressors. MiRNAs are involved in most cellular processes, and their expression and function are presided by several factors. Amongst, miRNA editing is an epitranscriptional modification that alters the original nucleotide sequence of selected miRNAs, possibly influencing their biogenesis and target-binding ability. A-to-I and C-to-U RNA editing are recognized as the canonical types, with the A-to-I type being the predominant one. Albeit some bioinformatics resources have been implemented to collect RNA editing data, it still lacks a comprehensive resource explicitly dedicated to miRNA editing. Here, we present MiREDiBase, a manually curated catalog of editing events in miRNAs. The current version includes 3,059 unique validated and putative editing sites from 626 pre-miRNAs in humans and three primates. Editing events in mature human miRNAs are supplied with miRNA-target predictions and enrichment analysis, while minimum free energy structures are inferred for edited pre-miRNAs. MiREDiBase represents a valuable tool for cell biology and biomedical research and will be continuously updated and expanded at https://ncrnaome.osumc.edu/miredibase.

m6A, one of the most abundant mRNA modifications, has been associated with various metabolic processes in plants. Here we show that m6A also plays a role in miRNA biogenesis in Arabidopsis thaliana. Significant reductions in plant m6A/MTA levels results in lower accumulation of miRNAs whereas pri-miRNA levels tend to be higher in such plants. m6A-IP Seq and MTA-GFP RIP were used to show that many pri-miRNAs are m6A methylated and are bound by MTA, further demonstrating that pri-miRNAs can also be substrates for m6A methylation by MTA. We report that MTA interacts with RNA Pol II, supporting the assumption that m6A methylation is a co-transcriptional process, and also identify TGH, a known miRNA biogenesis related protein, as a novel protein that interacts with MTA. Finally, reduced levels of miR393b may partially explain the strong auxin insensitivity seen in Arabidopsis plants with reduced m6A levels.

Background. miRNA regulates cellular processes by acting on specific target genes, and cellular processes proceed through multiple interactions often organized into pathways among genes and gene products. Hundreds of miRNAs and their target genes have been identified, as are many miRNA-disease associations. These, together with huge amounts of data on gene annotation, biological pathways, and protein-protein interactions are available in public databases. Here, using such data we built a database and web service platform, miRNA Disease Regulatory Network, or miRDRN, for users to construct disease and tissue-specific miRNA-protein regulatory networks, with which they may explore disease related molecular and pathway associations, or find new ones, and possibly discover new modes of drug action. Methods. Data on disease-miRNA association, miRNAtarget association and validation, gene-tissue association, gene-tumor association, biological pathways, human protein interaction, gene ID, gene ontology, gene annotation and product were collected from publicly available databases and integrated. A large set of miRNA target-specific regulatory sub-pathways (RSPs) having the form (T, G 1 , G 2) was built from the integrated data and stored, where T is a miRNA-associated target gene, G 1 (G 2) is a gene/protein interacting with T (G 1). Each sequence (T, G 1 , G 2) was assigned a p-value weighted by the participation of the three genes in molecular interactions and reaction pathways. Results. A web service platform, miRDRN (http://mirdrn.ncu.edu.tw/mirdrn/), was built. The database part of miRDRN currently stores 6,973,875 p-valued RSPs associated with 116 diseases in 78 tissue types built from 207 diseases-associated miRNA regulating 389 genes. miRDRN also provides facilities for the user to construct disease and tissue-specific miRNA regulatory networks from RSPs it stores, and to download and/or visualize parts or all of the product. User may use miRDRN to explore a single disease, or a disease-pair to gain insights on comorbidity. As demonstrations, miRDRN was applied: to

Coronary heart disease (CHD), as well as diabetes illness, remain the cause of mortality worldwide; hence, founding new biomarkers measurable in the initial stage of illness is necessary to accelerate treatment efficiency. Recently evidence has demonstrated alterations in miRNA levels associated with deregulated gene expression in diabetes and CHD. This case-control study investigates the probable effect of miR-146a rs2910164 C /G polymorphism in diabetes and atherosclerotic cardiovascular disease in Egyptian patients. The study comprised 210 individuals, including 70 CHD patients without diabetes, 70 CHD patients with diabetes, and 70 healthy controls, of Egyptian origin. Anthropometric and blood biochemical parameters were measured as well genetic analysis for rs2910164 C/G polymorphism was performed for all subjects using TaqMan realtime PCR assay. Our results revealed that; the biomedical parameters have a significant correlation between CHD with and without diabetes patients and healthy controls with a p-value <0.05. Analyses of genotype distribution for (rs2910164 C/G) revealed a significant association in both CHD with and without diabetes and control (odd ratio= 1.56, confidence interval (CI 95 %) = (0.06-0.63)) and (odd ratio= 0.88, (CI 95 %) = (0.83-0.92)) respectively. The current study's findings indicated that the selected polymorphism, miR-146a rs2910164 could represent a useful biomarker for susceptibility to CHD and diabetes in the Egyptian people.

The Zika virus (ZIKV) outbreak and spread is a global health emergency declared by World Health Organization. ZIKV rapidly spread across the world, causing neurological disorders. It is gaining public and scientific consideration. ZIKV genome biology and molecular structure are better understood with published papers. Genetic regulation is better understood by finding the motif in the DNA Genome sequence. The transcription factor binding sites need to be identified to understand the genetic code. There is diversity in gene expression. Motif-finding methods work towards efficiently identifying the repeated patterns in the genome. ZIKV genome sequence is used in the study. Identifying the motif is still a difficult task. There is a low probability of identifying the binding sites. Finding all possible solutions is challenging as it requires a lot of time and has high space complexity for finding long motifs. The Greedy search technique with pseudocount finds the motif in real-time. The count matrix is computed, and the profile matrix is constructed from the genome of the Zika virus. The calculated consensus string helps in calculating the score of the motif. The Greedy motif search technique is applied in this paper to find the motifs in the Zika virus Genome. This technique is not applied earlier to find the motifs in Zika Virus. The motifs are identified using a Greedy motif search without pseudocount and with pseudocount.

Background: Vascular invasion is closely related to the prognosis of hepatocellular carcinoma (HCC). Increasing evidence suggests that miRNAs can serve as biomarks to predict prognosis in various tumors. Thus, the aim of this study was to develop a novel, vascular invasion-related miRNA signature for prediction of HCC prognosis.Methods: Differentially expressed miRNAs (DEMs) between HCC samples with vascular invasion and without vascular invasion were obtained from GSE67140. MiRNAs expression profiles and clinical information for 344 patients were collected from The Cancer Genome Atlas database, and the patients were randomized (1:1) to training set and validation set. LASSO regression model was employed to identify survival-associated DEMs and establish risk score in the training set. Moreover, risk score was verified in the validation set. And nomogram based on risk score and clinical information was constructed to improve the prediction of prognosis. Meanwhile, four online tools ...

BACKGROUNDThe program of embryonic development is launched by selective activation of a silent maternal transcriptome. InCaenorhabditis elegans, nuclei of the adult germline are responsible for the synthesis of at least two distinct mRNA populations; those required for housekeeping functions, and those that program the oocyte-to-embryo transition. We mapped this separation by changes to the length-distribution of poly(A)-tails that depend on GLD-2 mediated cytoplasmic polyadenylation and its regulators genome-wide.RESULTSMore than 1000 targets of cytoplasmic polyadenylation were identified by differential polyadenylation. Amongst mRNA with the greatest dependence on GLD-2 were those encoding RNA binding proteins with known roles in spatiotemporal patterning such asmex-5andpos-1. In General, the 3’ UTR of GLD-2 targets were longer, contained cytosine-patches, and were enriched for non-standard polyadenylation-motifs. To identify the deadenylase that initiated transcript silencing, we...

Purpose Blue light has been reported to enhance the differentiation and mineralisation potential of mesenchymal stem cells. No studies have investigated the photobiomodulation effects of blue light on the odontogenic differentiation of dental pulp stem cell-containing populations. This study aimed to determine the effect of 405-nm wavelength on the mineralisation response in these cells. Methods Human dental pulp cells were irradiated using a calibrated 405 nm light source at an irradiance of 57.7 mW/cm2 for 35, 69, 90 and 104 s; delivering energy densities of 2, 4, 5 and 6 J/cm2. Cell proliferation was investigated on days 2 and 4. Alkaline phosphatase activity and dentine matrix protein-1 levels were investigated on days 3 and 5. Mineralised nodule formation was investigated on days 7, 4 and 21. Results The 405-nm blue light had significant inhibitory effects on dental pulp cell proliferation. Simultaneously, blue light irradiation enhanced the mineralisation response, as determin...

The outbreak of Zika virus (ZIKV) in the Americas has transformed a previously obscure mosquito-transmitted arbovirus of the Flaviviridae family into a major public health concern. Little is currently known about the evolution and biology of ZIKV and the factors that contribute to the associated pathogenesis. Determining genomic sequences of clinical viral isolates and characterization of elements within these are an important prerequisite to advance our understanding of viral replicative processes and virus-host interactions. We obtained a ZIKV isolate from a patient who presented with classical ZIKV-associated symptoms, and used high throughput sequencing and other molecular biology approaches to determine its full genome sequence, including non-coding regions. Genome regions were characterized and compared to the sequences of other isolates where available. Furthermore, we identified a subgenomic flavivirus RNA (sfRNA) in ZIKV-infected cells that has antagonist activity against R...

This study aimed to optimise techniques for whole transcriptome and small RNA analyses on clinical tissue samples from patients with cardiovascular disease. Clinical samples often represent a particular challenge to extracting RNA of sufficient quality for robust RNA sequencing analysis, and due to availability, it is rarely possible to optimise techniques on the samples themselves. Therefore, we have used equivalent samples from pigs undergoing cardiopulmonary bypass surgery to test different protocols for optimal RNA extraction, and then validated the protocols in human samples. Here we present an assessment of the quality and quantity of RNA obtained using a variety of commercially-available RNA extraction kits on both left ventricular biopsies and blood plasma. RNA extraction from these samples presents different difficulties; left ventricular biopsies are small and fibrous, while blood plasma has a low RNA content. We have validated our optimised extraction techniques on human clinical samples collected as part of the ARCADIA (Association of non-coding RNAs with Coronary Artery Disease and type 2 Diabetes) cohort study, resulting in successful whole transcriptome and small RNA sequencing of human left ventricular tissue.

MicroRNAs (miRNAs), a class of approximately 22 nucleotide long non coding RNAs which play critical role in different biological processes. The mature microRNA is usually 19–27 nucleotides long and is derived from a bigger precursor that folds into a flawed stem-loop structure. Mature micro RNAs are involved in many cellular processes that encompass development, proliferation, stress response, apoptosis, and fat metabolism by gene regulation. Resent finding reveals that certain viruses encode their own miRNA that processed by cellular RNAi machinery. In recent research indicate that cellular microRNA can target the genetic material of invading viruses. Cellular microRNA can be used in the virus life cycle; either to up regulate or down regulate viral gene expression Computational tools use in miRNA target prediction has been changing drastically in recent years. Many of the methods have been made available on the web and can be used by experimental researcher and scientist without e...

Objective: MicroRNAs are endogenous, small, single stranded, non coding RNAs having 19-25 nucleotides. These miRNAs are complementary to their target messenger RNAs that bind principally to its 3' un translated regions (3' UTRs). Small RNAs play crucial roles in the regulation of gene expression in many eukaryotes; therefore it is important to predict potential viral miRNAs which might be involved in an establishment of Japanese Encephalitis virus (JEV) disease. Different computational approaches and methods were used for predicting viral microRNAs from the JEV genome in this work. Methods: In the present study, the use of genome-wide computational approach has been demonstrated to predict miRNAs and their target(s) in JEV genome. Two freely accessible softwares, MiPred and Genscan were used to predict the secondary structures of the potential miRNAs. Results: In all, 36 miRNAs were predicted and characterized by conducting genome-wide homology search against all the reported miRNAs. These miRNAs were further validated by performing phylogenetic analyses and using statistical tools.Further, attempt was made to predict the 3′ untranslated regions of mRNAs from whole genome of JEV which may prove helpful in finding putative targets of these miRNAs. Conclusion: This is the first study to identify and validate miRNAs in JEV which is an important step in identifying putative JEV miRNAs that utilize host cell machinery, and may play a crucial role in neuroinflammation and silencing of host genes, thus demonstrating the role of viral miRNAs in establishing viral pathogenesis.

A gender gap exists in cystic fibrosis (CF). Here we investigate whether plasma microRNA expression profiles differ between the sexes in CF children. MicroRNA expression was quantified in paediatric CF plasma (n = 12; six females; Age range:1–6; Median Age: 3; 9 p.Phe508del homo- or heterozygotes) using TaqMan OpenArray Human miRNA Panels. Principal component analysis indicated differences in male versus female miRNA profiles. The miRNA array analysis revealed two miRNAs which were significantly increased in the female samples (miR-885-5p; fold change (FC):5.07, adjusted p value: 0.026 and miR-193a-5p; FC:2.6, adjusted p value: 0.031), although only miR-885-5p was validated as increased in females using specific qPCR assay (p &lt; 0.0001). Gene ontology analysis of miR-885-5p validated targets identified cell migration, motility and fibrosis as processes potentially affected, with RAC1-mediated signalling featuring significantly. There is a significant increase in miR-885-5p in plas...

† Background and Aims The clone EMB-2 of the interspecific hybrid Helianthus annuus  H. tuberosus provides an interesting system to study molecular and physiological aspects of somatic embryogenesis. Namely, in addition to non-epiphyllous (NEP) leaves that expand normally, EMB-2 produces epiphyllous (EP) leaves bearing embryos on the adaxial surface. This clone was used to investigate if the ectopic expression of H. annuus LEAFY COTYLEDON1-LIKE (Ha-L1L) gene and auxin activity are correlated with the establishment of embryogenic competence. † Methods Ha-L1L expression was evaluated by semi-quantitative RT-PCR and in situ hybridization. The endogenous level and spatial distribution of free indole-3-acetic acid (IAA) were estimated by a capillary gas chromatography-mass spectrometry-selected ion monitoring method and an immuno-cytochemical approach. † Key Results Ectopic expression of Ha-L1L was detected in specific cell domains of the adaxial epidermis of EP leaves prior to the development of ectopic embryos. Ha-L1L was expressed rapidly when NEP leaves were induced to regenerate somatic embryos by in vitro culture. Differences in auxin distribution pattern rather than in absolute level were observed between EP and A-2 leaves. More precisely, a strong IAA immuno-signal was detected in single cells or in small groups of cells along the epidermis of EP leaves and accompanied the early stages of embryo development. Changes in auxin level and distribution were observed in NEP leaves induced to regenerate by in vitro culture. Exogenous auxin treatments lightly influenced Ha-L1L transcript levels in spite of an enhancement of the regeneration frequency. † Conclusions In EP leaves, Ha-L1L activity marks the putative founder cells of ectopic embryos. Although the ectopic expression of Ha-L1L seems to be not directly mediated by auxin levels per se, it was demonstrated that localized Ha-L1L expression and IAA accumulation in leaf epidermis domains represent early events of somatic embryogenesis displayed by the epiphyllous EMB-2 clone.

Age-related changes in vascular functioning are a harbinger of cardiovascular disease but the biological mechanisms during the progression of endothelial senescence have not been studied. We investigated alterations in the proteome and miRNA profiles in the course of replicative senescence using primary human umbilical vein endothelial cells as an in vitro vascular model. Quantitative proteomic profiling from early growth stage to senescence was performed by isotope-coded protein label coupled to LC-ESI-MS/MS analysis. Some proteins consistently changed their expression during the senescence whereas others appeared as deregulated only during the late senescence. The latter was accompanied by alterations in morphology of senescent endothelial cells. MicroRNA expression profiling revealed transient changes in the level of miR-16-5p, miR-28-3p and miR-886-5p in the early senescence, decrease in the level of miR-106b-3p at the late stage, and continuous changes in the expression of miR-181a-5p and miR-376a-3p during the whole senescence process. Integrating data on proteomic and microRNA changes indicated potential crosstalk between specific proteins and non-coding RNAs in the regulation of metabolism, cell cycle progression and cytoskeletal organization in the endothelial senescence. The knowledge of molecular targets that change during the senescence can ultimately contribute to a better understanding and prevention of age-related vascular diseases.

MicroRNAs (miRNAs) are small non-coding RNAs that play an important role in the post-transcriptional regulation of gene expression. miRNAs are involved in the regulation of many biological processes such as differentiation, apoptosis, and cell proliferation. miRNAs are expressed in embryonic, postnatal, and adult hearts, and they have a key role in the regulation of gene expression during cardiovascular development and disease. Aberrant expression of miRNAs is associated with abnormal cardiac cell differentiation and dysfunction. Tbx5 is a member of the T-box gene family, which acts as transcription factor involved in the vertebrate heart development. Alteration of Tbx5 level affects the expression of hundreds of genes. Haploinsufficiency and gene duplication of Tbx5 are at the basis of the cardiac abnormalities associated with Holt-Oram syndrome (HOS). Recent data indicate that miRNAs might be an important part of the regulatory circuit through which Tbx5 controls heart development. Using high-throughput technologies, we characterized genome-widely the miRNA and mRNA expression profiles in WT-and Tbx5-depleted zebrafish embryos at two crucial developmental time points, 24 and 48 h post fertilization (hpf). We found that several miRNAs, which are potential effectors of Tbx5, are differentially expressed; some of them are already known to be involved in cardiac development and functions, such as miR-30, miR-34, miR-190, and miR-21. We performed an integrated analysis of miRNA expression data with gene expression profiles to refine computational target prediction approaches by means of the inversely correlation of miRNA-mRNA expressions, and we highlighted targets, which have roles in cardiac contractility, cardiomyocyte proliferation/apoptosis, and morphogenesis, crucial functions regulated by Tbx5. This approach allowed to discover complex regulatory circuits involving novel miRNAs and protein coding genes not considered before in the HOS such as miR-34a and miR-30 and their targets.

Microdeletion syndromes are genetic diseases caused by chromosomal deletions too small to be detected by karyotyping. They are typified by complex pleiotropic developmental phenotypes that depend both on the extent of the deletion and variations in genetic background. Microdeletion alleles disrupt several genes simultaneously, often as the result of a single mutagenic event, causing a wide array of consequences across multiple systems involving multiple pathways. How simultaneous haploinsufficiency of numerous adjacent genes leads to complex and variable pleiotropic phenotypes is not well understood. CRISPR/Cas9 genome editing has been shown to induce microdeletion-like alleles at a meaningful rate. Here, we describe a microdeletion allele in Caenorhabditis elegans recovered during a CRISPR/Cas9 genome editing experiment. We mapped the allele to chromosome V, balanced it with a reciprocal translocation crossover suppressor, and precisely defined the breakpoint junction. The allele simultaneously removes 32 protein-coding genes, yet animals homozygous for this mutation are viable as adults. Homozygous animals display a complex phenotype including maternal effect lethality, producing polynucleated embryos that grow into uterine tumors, vulva morphogenesis defects, body wall distensions, uncoordinated movement, and a shortened life span typified by death by bursting. Our work provides an opportunity to explore the complexity and penetrance of microdeletion phenotypes in a simple genetic model system.

Exclusive breastfeeding practice is relatively low among children below six months of age, despite the countless advantages provided by human milk. Improving and enhancing the immune system of the infants are important as their immune system is not completely developed yet in the early life. Breast milk or human milk is relatively abundant with bioactive components and nutrients and offers a complete source of food to the infants. The bioactive components of human milk are believed to provide infants with great protection. Currently, researchers discover the roles of microRNAs in human milk of humans towards proper immune system development of the infants. This systematic review aims to access and collect data on the diverse immunerelated microRNAs in the human milk and their immunoregulatory functions. To obtain related articles pertaining to the human milk microRNAs, a systematic search was conducted by using three relevant databases namely Science Direct, Scopus and PubMed using appropriate keywords for the papers published in English between 2010 and 2019. The included papers in this systematic review were appraised using Crowe Critical Appraisal Tool (CCAT). From 314 potential papers identified initially, five papers met the inclusion criteria for this review. All papers were of moderate to high quality. Discussion: This systematic review has provided valuable information on numerous immune-related microRNAs that can be found in human milk, including their immunological functions. The most abundantly expressed microRNAs include miR-146b, miR-148a, and miR-146a. The findings suggest that the microRNAs found in human milk along with their immunoregulatory functions were crucial in strengthening immune system of the infants.

Zika virus (ZIKV) is a serious public health concern that may lead to neurological disorders in affected individuals. The virus can be transmitted from an infected mother to her fetus, via mosquitoes, or sexually. ZIKV infections are associated with increased risk for Guillain-Barré syndrome (GBS) and congenital microcephaly in newborns infected prenatally. Dysregulations of intracellular microRNAs (miRNAs) in infected neurons have been linked to different neurological diseases. To determine the potential role of miRNAs in ZIKV infection we developed a chronically infected neuroblastoma cell line and carried out differential expression analyses of miRNAs with reference to an uninfected neuroblastoma cell line. A total of 3192miRNAs were evaluated and 389 were found to be upregulated < 2-fold and 1291 were downregulated < 2-fold. In particular, we determined that hsa-mir-431-5p, hsa-mir-3687, hsamir-4655-5p, hsa-mir-6071, hsa-mir-762, hsa-mir-5787, and hsa-mir-6825-3p were significantly downregulated, ranging from-5711 to-660-fold whereas, has-mir-4315, hsa-mir-5681b, hsamir-6511a-3p, hsa-mir-1264, hsa-mir-4418, hsa-mir-4497, hsa-mir-4485-3p, hsa-mir-4715-3p, hsamir-4433-3p, hsa-mir-4708-3p, hsa-mir-1973 and hsa-mir-564 were upregulated, ranging from 20-0.8-fold. We carried out target gene alignment of these miRNAs with the ZIKV genome to predict the function of the differentially expressed miRNAs and their potential impact on ZIKV pathogenesis. These miRNAs might prove useful as novel diagnostic or therapeutic markers and targets for further research on ZIKV infection and neuronal injury resulting from ZIKV infectivity in developing fetal brain neurons.

Up-regulation of Mirta22/Emc10 is a major transcriptional effect of the 22q11.2-associated microRNA dysregulation and underlies key cellular as well as behavioral deficits. EMC10 is a component of the ER membrane complex, which promotes membrane insertion of a subset of polytopic and tail-anchored membrane proteins. Here we show that EMC10 expression is elevated in hiPSC-derived neurons from 22q11.2 deletion carriers and that reduction of EMC10 levels restores defects in neurite outgrowth and calcium signaling. Moreover, using both a conditional genetic knock-out and injection of Antisense Oligonucleotides, we demonstrate that normalization of Emc10 levels in adult mouse brain rescues social memory deficits. The observations that elevated EMC10 expression is deleterious in 22q11.2 deletion carriers and that sustained elevation of EMC10 throughout the adult life can interfere with neural processes point to manipulations of EMC10 levels and downstream targets as a specific venue to am...

Genetic and epidemiological studies have found that variations in the amyloid precursor protein (APP) and the apoliopoprotein E (APOE) genes represent major modifiers of the progressive neurodegeneration in Alzheimer’s disease (AD). An extra copy or gain-of-function mutations inAPPlower age of AD onset. Compared to the other isoforms (APOE3andAPOE2), the ε4 allele ofAPOE(APOE4) hastens and exacerbates early and late onset forms of AD. Convenientin vivomodels to study how APP and APOE4 interact at the cellular and molecular level to influence neurodegeneration are lacking. Here, we show that the nematodeC. eleganscan model important aspects of AD including age-related, patterned neurodegeneration that is exacerbated byAPOE4. Specifically, we found thatAPOE4, but notAPOE3, acts withAPPto hasten and expand the pattern of cholinergic neurodegeneration caused byAPP. Molecular mechanisms underlying how APP and APOE4 synergize to kill some neurons while leaving others unaffected may be unc...

† Background and Aims The clone EMB-2 of the interspecific hybrid Helianthus annuus  H. tuberosus provides an interesting system to study molecular and physiological aspects of somatic embryogenesis. Namely, in addition to non-epiphyllous (NEP) leaves that expand normally, EMB-2 produces epiphyllous (EP) leaves bearing embryos on the adaxial surface. This clone was used to investigate if the ectopic expression of H. annuus LEAFY COTYLEDON1-LIKE (Ha-L1L) gene and auxin activity are correlated with the establishment of embryogenic competence. † Methods Ha-L1L expression was evaluated by semi-quantitative RT-PCR and in situ hybridization. The endogenous level and spatial distribution of free indole-3-acetic acid (IAA) were estimated by a capillary gas chromatography-mass spectrometry-selected ion monitoring method and an immuno-cytochemical approach. † Key Results Ectopic expression of Ha-L1L was detected in specific cell domains of the adaxial epidermis of EP leaves prior to the development of ectopic embryos. Ha-L1L was expressed rapidly when NEP leaves were induced to regenerate somatic embryos by in vitro culture. Differences in auxin distribution pattern rather than in absolute level were observed between EP and A-2 leaves. More precisely, a strong IAA immuno-signal was detected in single cells or in small groups of cells along the epidermis of EP leaves and accompanied the early stages of embryo development. Changes in auxin level and distribution were observed in NEP leaves induced to regenerate by in vitro culture. Exogenous auxin treatments lightly influenced Ha-L1L transcript levels in spite of an enhancement of the regeneration frequency. † Conclusions In EP leaves, Ha-L1L activity marks the putative founder cells of ectopic embryos. Although the ectopic expression of Ha-L1L seems to be not directly mediated by auxin levels per se, it was demonstrated that localized Ha-L1L expression and IAA accumulation in leaf epidermis domains represent early events of somatic embryogenesis displayed by the epiphyllous EMB-2 clone.

m 6 A, one of the most abundant mRNA modifications, has been associated with various metabolic processes in plants. Here we show that m 6 A also plays a role in miRNA biogenesis in Arabidopsis thaliana. Significant reductions in plant m 6 A/MTA levels results in lower accumulation of miRNAs whereas pri-miRNA levels tend to be higher in such plants. m 6 A-IP Seq and MTA-GFP RIP were used to show that many pri-miRNAs are m 6 A methylated and are bound by MTA, further demonstrating that pri-miRNAs can also be substrates for m 6 A methylation by MTA. We report that MTA interacts with RNA Pol II, supporting the assumption that m 6 A methylation is a co-transcriptional process, and also identify TGH, a known miRNA biogenesis related protein, as a novel protein that interacts with MTA. Finally, reduced levels of

The Zika virus (ZIKV) outbreak and spread is a global health emergency declared by World Health Organization. ZIKV rapidly spread across the world, causing neurological disorders. It is gaining public and scientific consideration. ZIKV genome biology and molecular structure are better understood with published papers. Genetic regulation is better understood by finding the motif in the DNA Genome sequence. The transcription factor binding sites need to be identified to understand the genetic code. There is diversity in gene expression. Motif-finding methods work towards efficiently identifying the repeated patterns in the genome. ZIKV genome sequence is used in the study. Identifying the motif is still a difficult task. There is a low probability of identifying the binding sites. Finding all possible solutions is challenging as it requires a lot of time and has high space complexity for finding long motifs. The Greedy search technique with pseudocount finds the motif in real-time. The count matrix is computed, and the profile matrix is constructed from the genome of the Zika virus. The calculated consensus string helps in calculating the score of the motif. The Greedy motif search technique is applied in this paper to find the motifs in the Zika virus Genome. This technique is not applied earlier to find the motifs in Zika Virus. The motifs are identified using a Greedy motif search without pseudocount and with pseudocount.

In the present study, we used high-throughput sequencing, miRNA-seq, to discover and explore the expression profiles of known and novel miRNAs in TLR ligand-stimulated vis-àvis non-stimulated (i.e. Control) peripheral blood mononuclear cells (PBMCs) isolated from blood of healthy Murrah buffaloes. Six small RNA (sRNA) libraries were multiplexed in Ion Torrent PI chip and sequenced on Ion Proton System. The reads obtained were aligned to the Bos taurus genome (UMD3.1 assembly), which is phylogenetically closest species to buffalo (Bubalus bubalis). A total of 160 bovine miRNAs were biocomputationally identified in buffalo PBMCs and 130 putatively novel miRNAs (not enlisted in the bovine mirBase) were identified. All of these 290 miRNAs identified across the six treatment and control samples represent the repertoire of novel miRNAs for the buffalo species. The expression profiles of these miRNAs across the samples have been represented by sample dendrogram and heatmap plots. The uniquely expressed miRNAs in each treatment and control groups were identified. A few miRNAs were expressed at very high levels while the majority of them were moderately expressed. The miRNAs bta-miR-103 and-191 were found to be highly abundant and expressed in all the samples. Other abundantly expressed miRNAs include bta-miR-19b,-29b,-15a,-19a,-30d,-30b-5p and members of let family (let 7a-5p, let 7g & let 7f) in LPS and CpG treated PBMCS and bta-miR-191,-103 &-19b in Poly I:C stimulated PBMCs. Only one novel miRNA (bta-miR-11039) out of 130 identified putatively novel miR-NAs, was expressed in all the six samples and differentially expressed (>2-fold) miRNAs were identified. Six of the differentially expressed miRNAs across the groups (bta-miR-421, bta-let-7i, bta-miR-138, bta-miR-21-5p, bta-miR-222 and bta-miR-27b) were subsequently confirmed by TaqMan quantitative reverse transcription polymerase chain reaction (qRT-PCR). Furthermore, the target genes of differentially expressed miRNAs were enriched for the roles in innate immunity and TLR signaling pathways. This maiden study on profiling and cataloguing of bubaline miRNAs expressed in TLR-ligand stimulated PBMCs will

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A gender gap exists in cystic fibrosis (CF). Here we investigate whether plasma microRNA expression profiles differ between the sexes in CF children. MicroRNA expression was quantified in paediatric CF plasma (n = 12; six females; Age range:1-6; Median Age: 3; 9 p.Phe508del homo-or heterozygotes) using TaqMan OpenArray Human miRNA Panels. Principal component analysis indicated differences in male versus female miRNA profiles. The miRNA array analysis revealed two miRNAs which were significantly increased in the female samples (miR-885-5p; fold change (FC):5.07, adjusted p value: 0.026 and miR-193a-5p; FC:2.6, adjusted p value: 0.031), although only miR-885-5p was validated as increased in females using specific qPCR assay (p < 0.0001). Gene ontology analysis of miR-885-5p validated targets identified cell migration, motility and fibrosis as processes potentially affected, with RAC1-mediated signalling featuring significantly. There is a significant increase in miR-885-5p in plasma of females versus males with CF under six years of age. Cystic fibrosis (CF) is an autosomal recessive genetic disorder arising from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene 1. CFTR is a crucial chloride ion channel expressed by epithelial cells in the lung and other organs. The consequences of defective CFTR are most severely manifested in the lung, where the imbalanced ion homeostasis results in chronic airway inflammation, which leads to a progressive and ultimately fatal decline in lung function. Despite improved therapies and increased life expectancy for both males and females with CF in recent years, a gender dichotomy persists whereby females are at a clinical disadvantage e.g. poorer lung function, earlier colonisation by chronic mucoid Pseudomonas aeruginosa, greater frequency of exacerbations, and lower median survival age 2-7. Whether sex differences exist in children with CF remains controversial 8,9. The ability to measure and monitor lung disease in children via a non-invasive method would be highly beneficial for CF clinical care. Molecular biomarkers that are prognostic for lung disease progression could serve, in support of clinical examination, to allow earlier and hence more effective intervention strategies. Ideally these would be molecules that are stable in body fluids (e.g. plasma), able to be quantified with reliance and accuracy, and mechanistically linked to pulmonary infection and/or inflammation. MicroRNAs are small non-coding RNAs that bind specific target mRNA sequences to inhibit their expression. As well as being expressed within cells/tissues, miRNAs are also present extracellularly, and are readily detectable in body fluids such as serum and plasma. In contrast to mRNA, miRNAs circulating in plasma are surprisingly resistant to RNase degradation due to the fact they are found either packaged inside microvesicles e.g. exosomes, or associated with protein/lipid complexes e.g. argonaute 2 or high density lipoprotein 10. Although the role of circulating miRNAs is not yet fully understood, their major function is thought to be cell-cell communication, for example between immune cells 10. It has been demonstrated that circulating miRNAs can be taken up by recipient cells and subsequently cause changes in target gene expression 11,12. Changes in circulating miRNA expression

Leishmaniaare intracellular protozoan parasites that cause a wide spectrum of clinical manifestations in genetically susceptible individuals with an insufficient or balanced Th1 immune response to eliminate the parasite. MiRNAs play important regulatory role in numerous biological processes including essential cellular functions. miR146-a acts as an inhibitor of interleukin 1 receptor associated kinase 1 (IRAK1) and tumour necrosis factor (TNF) receptor associated factor 6 (TRAF6) present in the toll-like receptors pathway while miR499a modulates TGF-β and TNF signalling pathways. Here, we investigated whetherMIRNA146A rs2910164 and MIRNA499 rs3746444variants are associated with the development ofL.guyanensis(Lg)-cutaneous leishmaniasis (CL). The variantsMIR146Ars2910164 andMIR499Ars3746444 were assessed in 850 patients withLg-CL and 891 healthy controls by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). Plasma cytokines were measured using the Bio...

In this study, we aimed to make a list of miRNA results from Nanostring analysis with prognostic strength, and then we intend to validate this crossing in an independent patient group. For this, we collected the HCC miRNA expression database using several data sets using Microarray, and then conducted a docking analysis to assess the prognostic potential to see the relationship between receptors and miRNA-targeted genes (Bcl2 / 5jsn).

MicroRNAs (miRNAs) are 19-25 nucleotides long, single-stranded, endogenous non-coding RNA molecules that play crucial roles in the post-transcriptional regulation of gene expression by targeting messenger RNAs for cleavage or translational repression. Genomes of various organisms ranging from higher animals and plants to viruses transcribe miRNAs. Recent studies show that both virus and host encode miRNAs that can give benefit either to virus or to host, depending upon the specific interactions. Hepatitis B Virus (HBV) is involved in acute and chronic diseases of liver and can bring about hepatitis, liver cirrhosis and hepatocellular carcinoma (HCC). In this study, we analyzed HBV (genotype D) for miRNAs, computationally, since computational methods serve vigorous, better, and affordable tools for microRNAs identification. Initial searches through VMir software extracted 57 sequences with potential hairpin-like structures from HBV genome. MiPred program confirmed 10 candidates as real pre-miRNA like hairpin structures. Measurements of free energy and other parameters further excluded three candidates. Finally, MatureBayes web server v1.0 confirmed 12 mature miRNAs in 6 potential pre-miRNA candidates in HBV genome, including one which has been identified in earlier studies. These findings open new avenues for researchers to explore the role of these novel miRNAs in viral pathogenesis as well as in developing novel antiviral therapies.

ACE2 is a transmembrane receptor located in cells in various tissues around the body. Its normal role is the conversion of Angiotensin II to Angiotensin 1-7 leading to vasodilation and a subsequent reduction in blood pressure via the renin-angiotensin-aldosterone system. ACE2 also plays a pivotal role in the infection of COVID-19 as it determines entry of virus into human cells. SARS-CoV-2 uses one of its four structural proteins, the spike (S) glycoprotein, to bind to the ACE2 receptor. This entry into the cell begins the process of infection and spread of the disease; because of the abundance of ACE2 throughout the body, viruses are able to enter a number of different organs. This multi-cell-entry via ACE2 is why COVID-19 affects multiple organ systems in the human body. Currently, therapies targeting ACE2 expression are limited. MicroRNA (miRNA)s are short non-coding RNA that downregulate the expression of a variety of proteins, and therefore offer a mechanism by which to inhibit...

Background: Numerous centrality measures have been introduced to identify "central" nodes in large networks. The availability of a wide range of measures for ranking influential nodes leaves the user to decide which measure may best suit the analysis of a given network. The choice of a suitable measure is furthermore complicated by the impact of the network topology on ranking influential nodes by centrality measures. To approach this problem systematically, we examined the centrality profile of nodes of yeast protein-protein interaction networks (PPINs) in order to detect which centrality measure is succeeding in predicting influential proteins. We studied how different topological network features are reflected in a large set of commonly used centrality measures. Results: We used yeast PPINs to compare 27 common of centrality measures. The measures characterize and assort influential nodes of the networks. We applied principal component analysis (PCA) and hierarchical clustering and found that the most informative measures depend on the network's topology. Interestingly, some measures had a high level of contribution in comparison to others in all PPINs, namely Latora closeness, Decay, Lin, Freeman closeness, Diffusion, Residual closeness and Average distance centralities. Conclusions: The choice of a suitable set of centrality measures is crucial for inferring important functional properties of a network. We concluded that undertaking data reduction using unsupervised machine learning methods helps to choose appropriate variables (centrality measures). Hence, we proposed identifying the contribution proportions of the centrality measures with PCA as a prerequisite step of network analysis before inferring functional consequences, e.g., essentiality of a node.

Developmental robustness relies on precise control of the timing and order of cellular events. In C. elegans, the invariant sequence of post-embryonic cell fate specification is controlled by oscillatory patterns of heterochronic microRNA transcription that are phase-locked with the larval molting cycle(1-4). How these transcriptional patterns are generated and how microRNA dosage is controlled is unknown. Here we show that transcriptional pulses of the lin-4 heterochronic microRNA are produced by two nuclear hormone receptors, NHR-85 and NHR-23, whose mammalian orthologs, Rev-Erb and ROR, function in the circadian clock. While Rev-Erb and ROR play antagonistic roles in regulating once-daily transcription(5-7), we find that NHR-85 and NHR-23 bind cooperatively as heterodimers to lin-4 regulatory elements to induce a single brief pulse of expression during each larval stage. We demonstrate that the timing and duration of lin-4 transcriptional pulses are programmed by the phased overl...

This study aimed to optimise techniques for whole transcriptome and small RNA analyses on clinical tissue samples from patients with cardiovascular disease. Clinical samples often represent a particular challenge to extracting RNA of sufficient quality for robust RNA sequencing analysis, and due to availability, it is rarely possible to optimise techniques on the samples themselves. Therefore, we have used equivalent samples from pigs undergoing cardiopulmonary bypass surgery to test different protocols for optimal RNA extraction, and then validated the protocols in human samples. Here we present an assessment of the quality and quantity of RNA obtained using a variety of commercially-available RNA extraction kits on both left ventricular biopsies and blood plasma. RNA extraction from these samples presents different difficulties; left ventricular biopsies are small and fibrous, while blood plasma has a low RNA content. We have validated our optimised extraction techniques on human clinical samples collected as part of the ARCADIA (Association of non-coding RNAs with Coronary Artery Disease and type 2 Diabetes) cohort study, resulting in successful whole transcriptome and small RNA sequencing of human left ventricular tissue.